NEW NMR METHODOLOGY FOR CHARACTERIZING CARBOHYDRATE-PROTEIN INTERACTIONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。碳水化合物-蛋白质复合体的形成在涉及细胞与其环境相互作用的各种过程中是重要的。这些过程包括自然的过程，如细胞分化、细胞聚集和细胞信号传递；它们还包括疾病过程，如病毒感染、恶性肿瘤和不必要的炎症。用来缓和自然过程和抑制疾病过程的药物的开发始于寡糖-蛋白质相互作用的准确结构模型。由于寡糖-蛋白质复合体中缺少短程NOE接触，这些模型很难用传统的核磁共振方法得到。我们正在开发一些实验和分析工具，允许确定方向约束，而不是在核磁共振研究中获得的传统距离约束。这些数据主要来自剩余的偶极偶联，当蛋白质和低聚糖在特定的场排列介质中被诱导定向时，这些偶极偶联变得可测量。必须收集这两个实体的数据，需要努力收集自然富含低聚糖的1H-13C对的数据，并指定大的或结构未确定的蛋白质中的1H-15N对。正在对中心内开发的分子系统以及外部合作者开发的系统进行开发测试。这些系统包括已知在炎症和恶性细胞转移中起重要作用的凝集素系统(Galectin-3，P-选择素)，以及已知在细胞表面碳水化合物合成中起重要作用的酶系统(LpxA，GnTV，ST6Gal1)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formation of carbohydrate-protein complexes is important in a variety of processes involving the interaction of a cell with its environment. These processes include natural ones such as cell differentiation, cell aggregation, and cell signaling; they also include disease processes such as viral infection, malignancy, and unwanted inflammation. The development of drugs to moderate natural processes and inhibit disease processes begins with accurate structural models for oligosaccharide-protein interactions. These models have been difficult to get by traditional Nuclear Magnetic Resonance (NMR) approaches because of the dearth of short-distance NOE contacts in oligosaccharide-protein complexes. We are developing a number of experiments and analysis tools that allow the determination of orientational constraints as opposed to the traditional distance constraints obtained in NMR studies. The data come largely from residual dipolar couplings that become measurable when protein and oligosaccharide are induced to orient in special field aligned media. Data must be collected on both entities, requiring efforts to deal with collection of data on 1H-13C pairs at natural abundance in oligosaccharides and assignment of 1H-15N pairs in large, or structurally uncharacterized, proteins. Developments are being tested on molecular systems developed within the center as well as systems developed by external collaborators. These include lectin systems (Galectin-3, P-selectin) known to be important in inflammation and in metastasis of malignant cells, as well as enzyme systems (LpxA, GnTV, ST6Gal1) known to be important in the synthesis of the cell surface carbohydrates.